Figure 1. (A) Work flow of the present study. A total of 25 consecutive patients undergoing carotid endarterectomy for AS, and 2 healthy subjects were enrolled. All patients underwent Doppler ultrasound imaging for the carotid plaques before endarterectomy, and the carotid atherosclerotic plaques were classified as complex lesion or “unstable” (UnS) and noncomplex or “stable” (S) plaques. Two UnS plaque samples were used for RNA-seq (one patient and one age-/gender matched healthy control), 5 for DNA methylation (2 patients and 2 healthy controls, including the same samples for RNA-seq, and bilateral carotid plaques from one same patient), 25 for protein and histological analysis (15 UnS and 10 S plaques), and 5 fresh samples to isolate CD14+ myeloid cells (i.e. monocytes and macrophages) for functional studies, including siRNA transfection and cytokines analysis for macrophages, and proliferation and cell cycle analysis for vascular smooth muscle cells (VSMCs). (B-E) Representative Doppler ultrasound images showing UnS (B, C) and S (D) carotid artery plaques, and a normal carotid artery (E). Plaques are demonstrated by red arrows.